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Preparation methods and basic principles of medium
The medium generally contains carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), vitamins and water. Artificial nutrients for the growth and maintenance of microbial, plant and animal tissues. The components of the unused medium are different, but the steps are the same.
Preparation of medium
1. Ingredients
Add a small amount of water (distilled water, natural water) to the container (distilled water, natural water). According to the formula, add all kinds of drugs (in turn) and fill the required amount of water (one tablespoon of medicine, close the bottle cap immediately after taking the medicine).
2. Dissolve
Starch dissolution: a small amount of cold water is blended into a paste
When heated and dissolved, especially the medium with Agar, it must be boiled. The melting temperature of Agar is 95 ℃ / 97 ℃, and it needs to be heated and agitated to prevent scorch.
3. Tune PH
Adjust the medium to the required value with 1N hydrochloric acid or 1N NaOH.
4. Filtration
Filter paper or cotton for filtering. (sometimes it can be omitted)
5. Subpackaging
The general medium is placed in a triangular flask or in a test tube for sterilization.
(1) Triangle bottle
If static culture was used, the triangle bottle of 100ml medium / 250ml could not exceed the triangle bottle of 150ml medium / 250ml at most, otherwise the culture medium boiling could easily pollute cotton cork and cause infection of bacteria in sterilized medium. If shake flask culture, then 15-20ml medium / 250ml triangle flask, to ensure good ventilation.
(2) in vitro assembly
The liquid culture medium is generally 4-5 ml, about 1 ~ 4 height of the test tube;
Solid slant culture medium is generally 3-4 ml, about 1-5 height of the test tube.
6. Bandage
After packing, plug cotton plug, wrap cotton plug with Kraft paper, prevent moisture from getting wet when sterilizing.
7. Sterilization
High pressure steam sterilization according to the temperature and pressure required in the formulation. If the temperature of sterilization is too high, nutrients will be destroyed, sugar in the medium, amino acids will make the color of the medium darker.
8. Pendulum plane
After sterilization, the test tubes that need to be tilted should be placed while they are hot, so that they solidify into a inclined surface, accounting for about 1 / 2 of the length of the test tube.
9. Storage
Medium placed at 30 ℃ for one day, no pollution can be used. Usually wrapped in Kraft paper and stored in 2? 8 ℃ refrigerator.
culture medium configuration principle
Select the appropriate nutrients
The growth and propagation of all microorganisms require the medium to contain carbon sources, nitrogen sources, inorganic salts, growth factors, water and energy, Therefore, a strong culture medium is firstly prepared according to the nutritional requirements of different microorganisms.
Proper concentration and proportion of nutrients
When the nutrient concentration in the medium is suitable, the microorganism can grow well, the nutrient concentration is too low to meet the needs of the normal growth of microorganism, and if the concentration is too high, it may inhibit the growth of the microorganism, such as the high concentration of sugar. Inorganic salts and heavy metal ions can not only not maintain and promote the growth of microorganisms, but also play an antibacterial or bactericidal role. In addition, the concentration ratio of nutrients in the medium also directly affected the growth and propagation of microorganisms and / or the formation and accumulation of metabolites, among which the ratio of carbon to nitrogen (C _ (N) was more important.
Control pH condition
The pH of the culture medium must be controlled within a certain range to satisfy the growth and reproduction of different types of microorganisms or the production of metabolites. In general, bacteria and actinomycetes are suitable for growth in the range of pH7-7.5, yeasts and molds usually grow in the range of pH4.5-6. It is worth noting that in the process of microbial growth, reproduction and metabolism, because of the decomposition and utilization of nutrients and the formation and accumulation of metabolites, the culture medium pH will change, and if the medium pH condition is not controlled, Often lead to microbial growth. Long-term decline or (and) decline in the production of metabolites. Therefore, in order to keep the medium pH relatively constant, pH buffer is usually added to the medium. The commonly used buffer is a mixture of monohydrogen and dihydrophosphate (such as KH2PO4 and K2HPO4).
Controlled redox potential (redox potential)
The growth of different types of microorganisms is different to the requirements of the oxidation reduction potential (F), and the general aerobic microorganisms can grow normally when the F value is + 0.1 V, and the anaerobic microorganisms can only grow under the condition that the F value is lower than + 0.1 V, The facultative anaerobic microorganism performs aerobic respiration when the F value is + 0.1 V or more, and performs fermentation at the time of + 0.1 V or less. The F-value is related to the oxygen partial pressure and the pH, and is also influenced by some of the microbial metabolites. Under the condition of relatively stable pH, the oxygen partial pressure of the culture medium can be increased by increasing the aeration amount (e.g., shaking culture, stirring), The reducing substances such as ascorbic acid, hydrogen sulfide, cysteine, glutathione, dithiothreonol and other reducing substances could decrease the F value.
5. Selection of raw materials
In the preparation of medium, cheap and easy-to-obtain raw materials should be used as the components of the medium, especially in the fermentation industry, the amount of culture medium is very large, and the use of low-cost raw materials shows its economic value.
biocidal treatment
In order to obtain the pure culture of the microorganism, it is necessary to avoid the contamination of the miscellaneous bacteria, so that the equipment and the working place used are sterilized and sterilized. In the case of the culture medium, the strict sterilization is to be carried out. The culture medium is generally sterilized by high-pressure steam, and the general culture medium is maintained for 15-30min under the condition of 1.05 kg/ cm2 and 121.3 & deg; C for 15-30min to achieve the purpose of sterilization.
The material comes from the network.
To learn more about biochemical reagents, please follow the official website: http://www.hnhbsj.com
(All contents of this website, where the source is the "Huibai reagent", the copyright is owned by the Huibai reagent, is not authorized, any media, website or individual shall not be reproduced, otherwise the legal liability will be held, and the "Source: Huibai reagent" shall be indicated in the authorization and transfer. The source of this network is the content of other media. The content of the other media is reprinted. The copyright is only shared by the original author. If the copyright is infringed, please contact us in time. )
Preparation of medium
1. Ingredients
Add a small amount of water (distilled water, natural water) to the container (distilled water, natural water). According to the formula, add all kinds of drugs (in turn) and fill the required amount of water (one tablespoon of medicine, close the bottle cap immediately after taking the medicine).
2. Dissolve
Starch dissolution: a small amount of cold water is blended into a paste
When heated and dissolved, especially the medium with Agar, it must be boiled. The melting temperature of Agar is 95 ℃ / 97 ℃, and it needs to be heated and agitated to prevent scorch.
3. Tune PH
Adjust the medium to the required value with 1N hydrochloric acid or 1N NaOH.
4. Filtration
Filter paper or cotton for filtering. (sometimes it can be omitted)
5. Subpackaging
The general medium is placed in a triangular flask or in a test tube for sterilization.
(1) Triangle bottle
If static culture was used, the triangle bottle of 100ml medium / 250ml could not exceed the triangle bottle of 150ml medium / 250ml at most, otherwise the culture medium boiling could easily pollute cotton cork and cause infection of bacteria in sterilized medium. If shake flask culture, then 15-20ml medium / 250ml triangle flask, to ensure good ventilation.
(2) in vitro assembly
The liquid culture medium is generally 4-5 ml, about 1 ~ 4 height of the test tube;
Solid slant culture medium is generally 3-4 ml, about 1-5 height of the test tube.
6. Bandage
After packing, plug cotton plug, wrap cotton plug with Kraft paper, prevent moisture from getting wet when sterilizing.
7. Sterilization
High pressure steam sterilization according to the temperature and pressure required in the formulation. If the temperature of sterilization is too high, nutrients will be destroyed, sugar in the medium, amino acids will make the color of the medium darker.
8. Pendulum plane
After sterilization, the test tubes that need to be tilted should be placed while they are hot, so that they solidify into a inclined surface, accounting for about 1 / 2 of the length of the test tube.
9. Storage
Medium placed at 30 ℃ for one day, no pollution can be used. Usually wrapped in Kraft paper and stored in 2? 8 ℃ refrigerator.
culture medium configuration principle
Select the appropriate nutrients
The growth and propagation of all microorganisms require the medium to contain carbon sources, nitrogen sources, inorganic salts, growth factors, water and energy, Therefore, a strong culture medium is firstly prepared according to the nutritional requirements of different microorganisms.
Proper concentration and proportion of nutrients
When the nutrient concentration in the medium is suitable, the microorganism can grow well, the nutrient concentration is too low to meet the needs of the normal growth of microorganism, and if the concentration is too high, it may inhibit the growth of the microorganism, such as the high concentration of sugar. Inorganic salts and heavy metal ions can not only not maintain and promote the growth of microorganisms, but also play an antibacterial or bactericidal role. In addition, the concentration ratio of nutrients in the medium also directly affected the growth and propagation of microorganisms and / or the formation and accumulation of metabolites, among which the ratio of carbon to nitrogen (C _ (N) was more important.
Control pH condition
The pH of the culture medium must be controlled within a certain range to satisfy the growth and reproduction of different types of microorganisms or the production of metabolites. In general, bacteria and actinomycetes are suitable for growth in the range of pH7-7.5, yeasts and molds usually grow in the range of pH4.5-6. It is worth noting that in the process of microbial growth, reproduction and metabolism, because of the decomposition and utilization of nutrients and the formation and accumulation of metabolites, the culture medium pH will change, and if the medium pH condition is not controlled, Often lead to microbial growth. Long-term decline or (and) decline in the production of metabolites. Therefore, in order to keep the medium pH relatively constant, pH buffer is usually added to the medium. The commonly used buffer is a mixture of monohydrogen and dihydrophosphate (such as KH2PO4 and K2HPO4).
Controlled redox potential (redox potential)
The growth of different types of microorganisms is different to the requirements of the oxidation reduction potential (F), and the general aerobic microorganisms can grow normally when the F value is + 0.1 V, and the anaerobic microorganisms can only grow under the condition that the F value is lower than + 0.1 V, The facultative anaerobic microorganism performs aerobic respiration when the F value is + 0.1 V or more, and performs fermentation at the time of + 0.1 V or less. The F-value is related to the oxygen partial pressure and the pH, and is also influenced by some of the microbial metabolites. Under the condition of relatively stable pH, the oxygen partial pressure of the culture medium can be increased by increasing the aeration amount (e.g., shaking culture, stirring), The reducing substances such as ascorbic acid, hydrogen sulfide, cysteine, glutathione, dithiothreonol and other reducing substances could decrease the F value.
5. Selection of raw materials
In the preparation of medium, cheap and easy-to-obtain raw materials should be used as the components of the medium, especially in the fermentation industry, the amount of culture medium is very large, and the use of low-cost raw materials shows its economic value.
biocidal treatment
In order to obtain the pure culture of the microorganism, it is necessary to avoid the contamination of the miscellaneous bacteria, so that the equipment and the working place used are sterilized and sterilized. In the case of the culture medium, the strict sterilization is to be carried out. The culture medium is generally sterilized by high-pressure steam, and the general culture medium is maintained for 15-30min under the condition of 1.05 kg/ cm2 and 121.3 & deg; C for 15-30min to achieve the purpose of sterilization.
The material comes from the network.
To learn more about biochemical reagents, please follow the official website: http://www.hnhbsj.com
(All contents of this website, where the source is the "Huibai reagent", the copyright is owned by the Huibai reagent, is not authorized, any media, website or individual shall not be reproduced, otherwise the legal liability will be held, and the "Source: Huibai reagent" shall be indicated in the authorization and transfer. The source of this network is the content of other media. The content of the other media is reprinted. The copyright is only shared by the original author. If the copyright is infringed, please contact us in time. )
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